The key to effective immunization is the generation of longlived memory T and B cells which have the capacity to provide rapid and effective secondary responses to the immunogen. The pathways by which memory T and B cells develop and the factors regulating the generation of memory T land B cells are largely unknown. We propose studies to examine the characteristics of CD4 memory, the pathway (s) for the generation of CD4 memory cells and the facts which regulate the size of the CD4 memory pool, its longevity and its effectiveness. One challenge to investigating memory is the fact that memory cells to a particular foreign Ag are rare and the use of single phenotypic markers for identifying memory populations is unreliable. We will use highly purified naive cells from T Cell Receptor transgenic mice to generate in vitro populations of effector cells that are virtually all Ag-specific and Ag- experienced. These populations can be studied both in vitro and after transfer to adoptive hosts, for progression to CD4 cells with memory phenotype and function. We can thus evaluate the facts which are likely to promote memory development, first in vitro where their mechanisms and detailed effects can be deduced, and then in vivo where their efficacy can be validated. We will focus on determining the role of Ag, cytokines and co-stimulatory molecules in promoting the expansion and longevity of CD4 effector cells and in driving effectors to express properties expected of a memory population. These include; (1) resistance to programmed cell death, (2) reversion to a small resting state, (3) expression of cell surface markers which characterize memory cells, (4) development of activation requirements characteristic of resting memory cells and (5) ability to produce a cytokine profile and function expected of a memory population. Effector and candidate pre-memory cell population will then be transferred to adoptive hosts to evaluate their in vivo potential to develop into bonafide memory cells. We will also evaluate the influence of Ag land key cytokines on the progression of the transferred populations to memory. These insights should highlight potential therapeutic approaches likely to optimize the generation of CD4 memory. This project will benefit greatly from the observations made and insights gained in the other projects. Project 2 Dutton) will pursue quite similar Aims for generation of CD8 memory. It will be particularly interesting to see if there are major differences between CD4 and CD8 cells, as might be expected by their different roles in immunity. With Linda Bradley (Project 3) we will examine the localization of effector populations we generate in vitro and the memory populations obtained after adoptive transfer. A key element of the development of memory may be the acquisition of the ability to recirculate independently of the L-selectin mediated extravasation through HEV. We will also collaborate extensively with Norem Klinman in determining the ability of the effector and memory populations we develop to drive the generation of B cell memory.